[Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae]. / Esporogénesis, esporodermo y ornamentación de esporas maduras en Lycopodiaceae.

Studies on reproductive aspects, spore morphology and ultrastructure of Lycopodiaceae are not very common in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. In order to complete existing information, adding new and broader contributions on these topics, a comparative analysis of the sporogenesis ultrastructure, with emphasis on cytological aspects of the sporocyte coat development, tapetum, monoplastidic and polyplastidic meiosis, sporoderm ontogeny and ornamentation of the mature spores, was carried out in 43 taxa of eight genera of the Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (including Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea and Pseudolycopodiella growing in the Andes of Colombia and the Neotropics. For this study, the transmission elec- tron microscopy (TEM) samples were collected in Cauca and Valle del Cauca Departments, while most of the spores for scanning electron microscopy (SEM) analysis were obtained from herbarium samples. We followed standard preparation procedures for spore observation by TEM and SEM. Results showed that the sporocyte coat is largely composed by primary wall components; the sporocyte develop much of their metabolic activity in the production of their coat, which is retained until the spores release; protective functions for the diploid cells undergoing meiosis is postulated here for this layer. The abundance of dictyosomes in the sporocyte cytoplasm was related to the formation and development of the sporocyte coat. Besides microtubule activity, the membrane of sporocyte folds, associated with electrodense material, and would early determine the final patterns of spore ornamentation. Monoplastidic condition is common in Lycopodium s.l., whereas polyplastidic condition was observed in species of Huperzia and Lycopodiella s. l. In monoplastidic species, the tapetum presents abun- dant multivesicular bodies, while in polyplastidic species, the secretory activity of the tapetum is less intense. Sporoderm development is centripetal, exospore is the first formed layer, then the endospore and, if present, perispore is the final deposited layer. Adult spores of the Lycopodiaceae showed two patterns of ornamentation: negative or caviform (foveolate spores) and positive or muriform ornamentation, the latter with two subtypes (rugate and reticulate spores). The spores of Huperzia are characteristically foveolate, the rugate spores were found in a few species of Huperzia and in all of the Lycopodiella s. l. taxa studied, while Lycopodium s.l. spores bear reticulate ornamentation. Numerous ornamentation traits are diagnostic at the specific level. The types of ornamentation found do not support the recent extreme fragmentation of the family in several genera, but could match, a priori, with the idea of three subfamilies. The findings of sporogenesis, extremely similar in all taxa studied, point more to consider fewer genera, more comprehensive, than the recent, marked splitting of the family.

Esporogénesis, esporodermo y ornamentaciónde esporas maduras en Lycopodiaceae / Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae

Studies on reproductive aspects, spore morphology and ultrastructure of Lycopodiaceae are not very common in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. In order to complete existing information, adding new and broader contributions on these topics, a comparative analysis of the sporogenesis ultrastructure, with emphasis on cytological aspects of the sporocyte coat development, tapetum, monoplastidic and polyplastidic meiosis, sporoderm ontogeny and ornamentation of the mature spores, was carried out in 43 taxa of eight genera of the Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (including Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea and Pseudolycopodiella growing in the Andes of Colombia and the Neotropics. For this study, the transmission electron microscopy (TEM) samples were collected in Cauca and Valle del Cauca Departments, while most of the spores for scanning electron microscopy (SEM) analysis were obtained from herbarium samples. We followed standard preparation procedures for spore observation by TEM and SEM. Results showed that the sporocyte coat is largely composed by primary wall components; the sporocyte develop much of their metabolic activity in the production of their coat, which is retained until the spores release; protective functions for the diploid cells undergoing meiosis is postulated here for this layer. The abundance of dictyosomes in the sporocyte cytoplasm was related to the formation and development of the sporocyte coat. Besides microtubule activity, the membrane of sporocyte folds, associated with electrodense material, and would early determine the final patterns of spore ornamentation. Monoplastidic condition is common in Lycopodium s.l., whereas polyplastidic condition was observed in species of Huperzia and Lycopodiella s. l.. In monoplastidic species, the tapetum presents abundant multivesicular bodies, while in polyplastidic species, the secretory activity of the tapetum is less intense. Sporoderm development is centripetal, exospore is the first formed layer, then the endospore and, if present, perispore is the final deposited layer. Adult spores of the Lycopodiaceae showed two patterns of ornamentation: negative or caviform (foveolate spores) and positive or muriform ornamentation, the latter with two subtypes (rugate and reticulate spores). The spores of Huperzia are characteristically foveolate, the rugate spores were found in a few species of Huperzia and in all of the Lycopodiella s. l. taxa studied, while Lycopodium s.l. spores bear reticulate ornamentation. Numerous ornamentation traits are diagnostic at the specific level. The types of ornamentation found do not support the recent extreme fragmentation of the family in several genera, but could match, a priori, with the idea of three subfamilies. The findings of sporogenesis, extremely similar in all taxa studied, point more to consider fewer genera, more comprehensive, than the recent, markedsplitting of the family. Rev. Biol. Trop. 62 (3): 1161-1195. Epub 2014 September 01.

Estudios sobre aspectos reproductivos, morfología y ultraestructura de las esporas de Lycopodiaceae no son abundantes en la literatura científica y constituyen información esencial para apoyar las relaciones taxonómicas y sistemáticas en el grupo. Con el fin de completar la información existente, añadiendo contribuciones nuevas y más amplias sobre estos temas, se realizó un análisis comparado de la ultraestructura de la esporogénesis, con énfasis en aspectos citológicos que tienen que ver con la formación de la cubierta de los esporocitos, el tapete, las meiosis monoplastidial y poliplastidial, la ontogenia del esporodermo y la ornamentación de las esporas maduras en 43 táxones de ocho géneros de Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (incluyendo Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea y Pseudolycopodiella que crecen en los Andes de Colombia y el Neotrópico. Para estudios con microscopía electrónica de trasmisión (MET) las muestras se recolectaron en los departamentos de Cauca y Valle del Cauca, mientras que la mayoría de las muestras para microscopía electrónica de barrido (MEB) provienen de material herborizado de colecciones. Para la observación de las muestras con MET y MEB se utilizaron protocolos estándar para el procesamiento de esporas. La cubierta de los esporocitos está formada por pared primaria; los esporocitos invierten gran parte de su actividad metabólica en la producción de esa cubierta, que es mantenida hasta la liberación de las esporas y tiene funciones de protección de las células que harán meiosis. La abundancia de dictiosomas en los esporocitos se relacionó con la formación y desarrollo de la cubierta. Además de la actividad de los microtúbulos, la presencia de sinuosidades y plegamientos asociados con material electro denso en la membrana de los esporocitos determinarían tempranamente los patrones de ornamentación de las esporas. La condición monoplastidial es común en Lycopodium s.l.y la poliplastidial se observó en Huperzia y Lycopodiella s. l. En especies monoplastidiales el tapete presenta abundantes cuerpos plurivesiculares, en las poliplastidiales la actividad secretora del tapete es menos intensa. El desarrollo del esporodermo es centrípeto, el exosporio se forma primero, seguido del endosporio y el perisporio, si está presente, se deposita de último. En las esporas adultas de Lycopodiaceae se encontraron dos patrones de ornamentación: negativo o caviforme (esporas foveoladas) y positivo o muriforme (esporas rugadas y reticuladas). Las esporas foveoladas son características de Huperzia; las rugadas de unas pocas especies de Huperzia y las especies de Lycopodiella s. l., mientras que las reticulada son típicas de Lycopodium s. l.. Numerosos caracteres de la ornamentación resultan diagnósticos en el nivel específico. Los tipos principales no apoyan la extrema fragmentación reciente de la familia en varios géneros, aunque podría coincidir, a priori, con la idea de tres subfamilias. Los hallazgos de la esporogénesis, extremadamente similar en todos los táxones estudiados, apuntan más a la unificación de los géneros en la familia que a su segregación.

[Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia]. / Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de Colombia.

Studies on some reproductive traits in Equisetum species are scarce and valuable to understand species distribution. Therefore, a detailed study of the sporogenesis process and spore development in E. bogotense is presented, with an analysis of the main events during meiosis, maturation of spores, spore wall ultrastructure, orbicules and elaters. Specimens were collected from 500 to 4500 m in Cauca, Colombia. Strobili at different maturation stages were fixed, dehydrated, embedded in resin, and ultra-microtome obtained sections were stained with Toluidine blue. Observations were made with optical microscopy with differential interference contrast illumination technique (DIC), transmission and scanning electron microscopy (TEM and SEM). Ultrathin sections (70-80 microm) for TEM observations were stained with uranyl acetate and lead citrate; while samples for SEM observations, were fixed, dehydrated in 2.2-dimethoxypropane and dried at critical point as in standard methods. Strobili have numerous mature sporangiophores, each one with a peltate structure, the scutellum, bearing five-six sessile sporangia attached to the axis of strobilus by the manubrium. Immature sporocytes (spore mother cells) are tightly packed within the young sporangia. The sporocytes quickly undergo meiosis, by passing the stage of archesporium and give origin to tetrads of spores. The tapetum loses histological integrity during early stages of sporogenesis, intrudes as a plasmodial mass into the cavity of the sporangium, partially surrounding premeiotic sporocytes, and then, tetrads and adult spores. The tapetum disintegrates towards the end of the sporogenesis, leaving spores free within the sporangial cavity. Spores present several cytological changes that allow them to achieve greater size and increase the number of plastids, before reaching the adult stage. Sporoderm includes three layers external to the cytoplasmic membrane of the spore cell, and they are pseudoendospore, exospore and perispore. Viewed with SEM, the exospore is smooth to rugulate, with micro perforations, while the perispore is muriform, rugate, with narrow, delicate, discontinuous, randomly distributed folds delimiting incomplete, irregular areolae, externally covered by of different size, densely distributed orbicules. These orbicules are also found all over the external face and margins of the elaters, while the internal face is smooth and lack orbicules. Viewed with TEM, the exospore is a thick layer of fine granular material, while perispore is a thinner layer of dense, separate orbicules. The elaters are composed by two layers of fibrillar material: an inner layer with longitudinally oriented fibrils and an outer, thicker and less dense layer with fibrils transversely fibrils and abundant, external orbicules. It is suggested that the processes of ontogeny and characters of the sporoderm are relatively constant in Equisetum; however, sporogenesis in E. bogotense is synchronous and this condition has been observed so far only in E. giganteum, a tropical genus also found in Colombia.

Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de Colombia / Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia

Studies on some reproductive traits in Equisetum species are scarce and valuable to understand species distribution. Therefore, a detailed study of the sporogenesis process and spore development in E. bogotense is presented, with an analysis of the main events during meiosis, maturation of spores, spore wall ultrastructure, orbicules and elaters. Specimens were collected from 500 to 4 500m in Cauca, Colombia. Strobili at different maturation stages were fixed, dehydrated, embedded in resin, and ultra-microtome obtained sections were stained with Toluidine blue. Observations were made with optical microscopy with differential interference contrast illumination technique (DIC), transmission and scanning electron microscopy (TEM and SEM). Ultrathin sections (70-80μm) for TEM observations were stained with uranyl acetate and lead citrate; while samples for SEM observations, were fixed, dehydrated in 2.2-dimethoxypropane and dried at critical point as in standard methods. Strobili have numerous mature sporangiophores, each one with a peltate structure, the scutellum, bearing five-six sessile sporangia attached to the axis of strobilus by the manubrium. Immature sporocytes (spore mother cells) are tightly packed within the young sporangia. The sporocytes quickly undergo meiosis, by passing the stage of archesporium and give origin to tetrads of spores. The tapetum loses histological integrity during early stages of sporogenesis, intrudes as a plasmodial mass into the cavity of the sporangium, partially surrounding premeiotic sporocytes, and then, tetrads and adult spores. The tapetum disintegrates towards the end of the sporogenesis, leaving spores free within the sporangial cavity. Spores present several cytological changes that allow them to achieve greater size and increase the number of plastids, before reaching the adult stage. Sporoderm includes three layers external to the cytoplasmic membrane of the spore cell, and they are pseudoendospore, exospore and perispore. Viewed with SEM, the exospore is smooth to rugulate, with micro perforations, while the perispore is muriform, rugate, with narrow, delicate, discontinuous, randomly distributed folds delimiting incomplete, irregular areolae, externally covered by of different size, densely distributed orbicules. These orbicules are also found all over the external face and margins of the elaters, while the internal face is smooth and lack orbicules. Viewed with TEM, the exospore is a thick layer of fine granular material, while perispore is a thinner layer of dense, separate orbicules. The elaters are composed by two layers of fibrillar material: an inner layer with longitudinally oriented fibrils and an outer, thicker and less dense layer with fibrils transversely fibrils and abundant, external orbicules. It is suggested that the processes of ontogeny and characters of the sporoderm are relatively constant in Equisetum; however, sporogenesis in E. bogotense is synchronous and this condition has been observed so far only in E. giganteum, a tropical genus also found in Colombia.

Los estudios sobre aspectos reproductivos son escasos en Equisetum. Por eso, hemos realizado un análisis detallado del proceso de esporogénesis, desarrollo de las esporas, ultraestructura de procesos que tienen lugar durante la meiosis, formación de la pared esporal, orbículas y eláteres de E. bogotense, en especímenes procedentes del Cauca, Colombia. Los estudios se efectuaron mediante microscopía fotónica, electrónica de transmisión (TEM) y de barrido (SEM). Los estróbilos llevan numerosos esporangióforos maduros, cada uno con un escutelo peltado, unido al eje del estróbilo por el manubrio y portador de 5-6 esporangios sésiles. Los esporocitos experimentan meiosis dando origen a tétradas de esporas. El tapete pierde la integridad histológica en las primeras etapas de esporogénesis y rodea los esporocitos premeióticos, posteriormente a las tétradas y finalmente las esporas inmaduras, que experimentan cambios citológicos y de tamaño antes de alcanzar la etapa adulta. El esporodermo de las esporas adultas de E. bogotense consiste de seudoendosporio, exosporio y perisporio. Vistos con MEB, el exosporio de las esporas adultas es liso a rugulado con microperforaciones y el perisporio es muriforme, rugado, con pliegues delicados, estrechos, discontinuos, que se distribuyen al azar y delimitan aréolas incompletas. Externamente el perisporio está cubierto por orbículas, que se forman también en la cara externa y los márgenes de los eláteres. Vistos con TEM, el exosporio es una capa de material granular fino y el perisporio, una capa mucho más delgada con orbículas discretas. Los eláteres están formados por dos capas de naturaleza fibrilar, orientadas longitudinalmente y transversalmente. La esporogénesis en E. bogotense es sincrónica, similar a la de E. giganteum, otra especie de distribución tropical que también crece en Colombia.

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia: interpolation of progenesis and asexual heterospory in an artificial sporangium.

Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23-25 °C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

Female parthenogenetic apomixis and androsporogenesis in Douglas-fir embryonal initials in an artificial sporangium.

Control of female parthenogenetic apomixis and androsporogenesis of Douglas-fir embryonal initials was studied using an experimental culture system in which changes in growth condition can mediate changes in cell identity and outcomes. This culture system constitutes an artificial sporangium in which myriad culture conditions can be simulated and should be applicable for research on a variety of gymnosperms. In this study, embryonal initials from developing seeds from two Douglas-fir trees were rescued and became reprogrammed for female parthenogenetic apomixis (fPA) and parthenogenetic androsporogenesis (mPA). Female PA was initiated by endomitosis forming a binucleate cell with a diploid egg-equivalent and an apoptotic ventral canal nucleus in an archegonial tube. Egg-equivalent nuclei formed cells (parthenotes) that were discharged into an aqueous culture medium. Parthenotes developed axial tiers atypical of early embryogenesis in seeds. Earlier in the year, androsporangial tubes were parthenogenetically differentiated and released monads, dyads, triads, and tetrads into the culture medium. Spores showed chromosomal aberrations. PA demonstrated a temporal separation in gender expression (dichogamy). Embryonal initials brought forward and by-passed the long juvenile phases normally needed for cells to develop into trees and express reproductive maturity. Expressions of fPA and mPA indicated that the specialized culture flasks served as an artificial sporangium (AS). Awareness is raised for the value of an AS for research in gymnosperm life cycles and as a teaching and research laboratory.